title: Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber.
abstract: We have isolated a full length 5-LOX cDNA clone from potato cDNA library using degenerate primers designed from conserved sequences of LOXs. Sequence analysis and comparison of the deduced amino acid sequence revealed high homology to other plant LOXs. We have expressed the cDNA in Escherichia coli and purified the recombinant protein to electrophoretic homogeneity by anion exchange liquid chromatography followed by HPLC on a Mono-Q column. Substrate specificity of the purified recombinant protein revealed LOX activity towards linoleic, linolenic acid, arachidonic acids as substrates with linoleic acid being the best substrate. The relative LOX activity as well as the product profiles for the recombinant L1 5-LOX are comparable to values determined for the purified potato tuber 5-LOX. When the recombinant L1 5-LOX and the native peak-2 5-LOX (the most abundant isozyme) were compared on SDS-PAGE, single bands of apparently identical mass 97,000 Da, was observed, which agrees well with the L1 molecular mass calculated from amino acid sequences.
authors.affiliation: Department of Veterinary Science, Pennsylvania State University, University Park 16802, USA.
authors.forename: X
authors.initials: X
authors.lastname: Chen
authors.forename: P
authors.initials: P
authors.lastname: Reddanna
authors.forename: G R
authors.initials: GR
authors.lastname: Reddy
authors.forename: R
authors.initials: R
authors.lastname: Kidd
authors.forename: G
authors.initials: G
authors.lastname: Hildenbrandt
authors.forename: C C
authors.initials: CC
authors.lastname: Reddy
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D005231:Fatty Acids, Unsaturated; D010940:Plant Proteins; D011994:Recombinant Proteins; D001094:Arachidonate 5-Lipoxygenase